1. Field of the Invention
This invention relates to a method for enhancing the photosynthetic activity and growth of a higher plant to increase its harvest yield and/or to enable its earlier harvest.
2. Description of Related Art
The development of a recombinant DNA technique has realized the incorporation of a certain exogenous gene into a higher plant and expression regulation of an existing gene therein. Only few experiments has been attempted to improve the characteristics concerning food production, such as the production yield of serials and crops. Recently, incorporation of a gene, coding enzyme participating in photosynthesis or carbohydrate metabolism, was achieved. Such gene was incorporated in anti-sense direction to inhibit the expression of the gene. The results indicated functional importance of the enzyme as a rate-determining factor of photosynthesis or carbohydrate metabolism. A Researcher in Germany have played a major role in the research.
Despite of it, no attempts have been performed on phenotypic expression of a certain gene in a higher plant using recombinant DNA technique to enhance the photosynthesis, which is a primary metabolism of a higher plant, and to improve its growth.
The object of this invention is to achieve phenotypic expression of a certain gene in a higher plant using recombinant DNA technique to enhance the photosynthesis, which is primary metabolism of a higher plant, to improve the crop productivity and yield potentiality and/or to enable the earlier harvest of the crop.
This invention provides a method for improving the productivity of a higher plant having chloroplasts by the phenotypic expression of cyanobacterial fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase in the chloroplasts.
Moreover, this invention provides a transgenic plant comprising a higher plant with a DNA fragment incorporated therein, the DNA fragment containing a base sequence coding cyanobacterial fructose-1,6-bisphosphatase/sedoheptulose- 1,7-bisphosphatase.
The fructose-1,6-bisphosphatase (FBPase) and sedoheptulose-1,7-bisphosphatase (SBPase) in the chloroplasts of a higher plant are the key (rate determining) enzymes of a photosynthetic reductive carbon system. The activities of these enzymes are regulated by photoreduction-potentiality. On the other hand, fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase (FBPase/SBPase), derived from Cyanobacterium synechococcus PCC 7942 gene, if found widely in a specific type of a prokaryotic algaexe2x80x94a Cyanobacterium. The primary structure and enzyme properties of cyanobacterial FBPase/SBPase are different from those of the FBPase or SBPase found in the chloroplasts of a higher plant. In addition, cyanobacterial FBPase/SBPase is composed of one protein, that is, a bi-functional enzyme exhibiting two kinds of enzyme activities, FBPase and SBPase.
FBPase-I, derived from Cyanobacterium synechococcus PCC 7942 gene, is a tetramer consisting of four subunits of 40 kDa identical with each other. After the treatment by 1 mM H2O2, the purified enzyme retained more than 80 % of native enzyme activity. The enzyme activity of FBPase-I was inhibited by AMP (Ki=0.26 mM), which is a specific inhibitor of cytoplasm-type FBPase. However, it was not inhibited by fructose-2,6-P2. The optimum pH for the enzyme activity was 8.0 and pI value of the enzyme was 4.8. FBPase-I hydrolyzed not only fructose-1,6-bisphosphate (Fru1,6-P2), but also sedoheptulose-1,7-bisphosphate (Sed1,7-P2). The activities of the purified enzyme for Fru1,6-P2 and Sed1,7-P2 were 11.7 xcexcmol/min/mg protein and 12.1 xcexcmol/min/mg protein, respectively. The Km values for Fru1,6-P2 and Sed1,7-P2 were 52 xcexcM and 118 xcexcM, respectively. The enzyme activity was proved to be dependent on Mg2+ concentration, also equally to typical FBPase. The dose-response curve showed sigmoidal curve equally to plastid FBPase, and the S0.5 value was shown to be 1.4xc2x10.1 mM. This enzyme itself was described in xe2x80x9cArchives of Biochemistry and Biophysics, Vol. 334, No. 1, pp. 27 to 36, 1996: Molecular characterization and resistance to hydrogen peroxide of two fructose-1,6-bisphosphatase from synechococcus PCC 7942xe2x80x9d.
The inventor incorporated fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase, isolated from Cyanobacterium synechococcus PCC 7942, into a tobacco plant so that the expressed protein was transferred to its chloroplasts. The FBPase activity, the SBPase activity and the photosynthetic ability of the transgenic plant were compared to those of the wild type strain. The results measured 7 weeks after seeding showed significant increase of these activities in the transgenic plant. Furthermore, after certain period of cultivation, the plant bodies of the transgenic plant proved to be taller than those of the wild type strain. In the transgenic plant, the areas of the blades, the diameters of the stalks, and the numbers and lengths of the roots were larger than those in the wild type strain. In addition, the contents of hexose, sucrose and starch were proved to be increased in blades, stalks and roots of the transgenic plant, compared with those of the wild type strain.
Accordingly, the photosynthetic ability of the transgenic plant, obtained by incorporation of cyanobacterial FBPase/SBPase into a tobacco plant, was improved. As the result, the ability of the transgenic plant to synthesize carbohydrate and starch is increased, and the growth was enhanced, indicating the increase of final anabolism of the transgenic plant. Therefore, incorporation of cyanobacterial FBPase/SBPase into the chloroplasts of a higher plant was proved to be a very effective technique for producing rareripe or high-yield plants.
The effect might be explained as follows. Triggered by environmental stresses, light and oxygen toxicity causes various kinds of injuries to plant bodies, resulting in a critical and limiting factor of food production. Contrary to FBPase and SBPase derived from a higher plant, cyanobacterial FBPase/SBPase is resistant against oxygen injury and thus considered to function under various environmental stresses. Moreover, a gene encoding the cyanobacterial FBPase/SBPase does not exist in higher plants, thereby eliminating the possibility of adverse effects by gene silencing.
In this invention, a vector to produce a recombinant DNA includes plasmids pBI101, pIN19 and pMSH-1. A wide variety of useful cultivated plants and woods capable of photosynthesis can be adopted as a higher plant in which the inventive recombinant DNA is incorporated. For example, the invention may be applied to serials such as maize, rice, wheat, barley, oat wheat, millet and barnyard millet, beans such as soy bean, vegetables such as potato and tomato, useful cultivated plants such as coleseed, cotton and tobacco, and trees.
An amino acid sequence may be deleted from or added to the amino acid sequence of sequence number 1, or a part of the sequence of the sequence number 1 may be substituted with another amino acid sequence in the scope of this invention, so far as the resulting peptide retains its enzymatic activity properties as fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphasphatase. Preferably, not lower than 85 percent, more preferably not lower than 95 percent of the amino acid sequence may be overlapped or identical with the amino acid sequence of the sequence number 1.
In a base sequence of sequence number 2, a base sequence referred to as base numbers from 1 to 1068 is essential for this invention, because this base sequence corresponds to a structural gene portion, that is, an amino acid sequence of the sequence number 1. In addition, a base sequence referred to as base numbers from xe2x88x92180 to 1170 is the most preferred embodiment of this invention.
These and other features and advantages of this invention will become apparent upon a reading of the detailed description and drawings.